Insulin increases glucose uptake into fat and muscle by modulating the number of GLUT4 glucose transporters on the surface of these cells. In the absence of insulin, GLUT4 is predominantly sequestered intracellularly by a bipartite, dynamic retention mechanism that involves slow exocytosis from intracellular compartments and rapid retrieval from the plasma membrane. Insulin induces a reversible recruitment of GLUT4 to the cell surface by altering the dynamic distribution of GLUT4 between the interior and cell surface. Regulation of the amount of GLUT4 on the plasma membrane is critical for regulation of whole body glucose homeostasis, with the increase in insulin-stimulated conditions counteracting hyperglycemia, and the exclusion from the plasma membrane of basal cells protecting against hypoglycemia. Insulin does not properly regulate GLUT4 trafficking in individuals with Type 2 diabetes. The molecular defect(s) underlying this insensitivity to insulin are not known. A more complete understanding of GLUT4 internalization may lead to the identification of novel targets for the development of pharmacologic intervention in the treatment of Type 2 diabetes. Although both endocytosis and exocytosis are important in determining the amount of GLUT4 on the cell surface in all conditions, the majority of past studies have focused on characterizing exocytosis, and therefore relatively little is know about the mechanisms that control GLUT4 internalization in basal conditions, in the presence of insulin, in the return to basal retention following stimulation, or how the insulin signal is transmitted to GLUT4 endocytosis. The objective of this application is to use quantitative biochemical and microscopy assays to characterize GLUT4 endocytosis, the results of which will significantly extend our understanding of GLUT4 translocation and insulin action at a molecular level. There are three specific aims in this project. 1) To use quantitative fluorescence microscopy and quantitative biochemical methods to characterize the internalization of GLUT4 in basal and insulin-treated adipocytes. These results will provide the conceptual foundation for the studies in the other aims. 2) To analyze the internalization kinetics of GLUT4 in cells in which insulin-signal transduction has been perturbed by the pharmacologic treatments, expression of dominant-interfering mutants and by small interfering RNA targeted protein knockdown. These results will provide novel information on the mechanisms of insulin regulation of endocytosis. 3) To map in studies of GLUT4 mutants the structural determinants that control internalization in basal and insulin-stimulated conditions. The results of these studies will provide a more complete molecular description of GLUT4 internalization, and thereby provide the necessary framework for understanding insulin-regulation of endocytosis at a molecular level.